This invention relates to novel mutant protease enzymes or enzyme variants useful in formulating detergent compositions and exhibiting improved wash performance in detergents; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM(copyright) (Novo Nordisk A/S), RELASE(copyright) (Novo Nordisk A/S), MAXAPEM(copyright) (Gist-Brocades N.V.), PURAFECT(copyright) (Genencor International, Inc.).
Further a number of protease variants are describe in the art, such as in EP 130756 (GENENTECH)(corresponding to U.S. Reissue Pat. No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) Nature 318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193 803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER and GAMBLE COMPANY); WO 95/30010 (PROCTER and GAMBLE COMPANY); WO 95/29979 (PROCTER and GAMBLE COMPANY); U.S. Pat. No. 5,543,302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A/S); WO 91/00345 (NOVO NORDISK A/S); EP 525 610 Al (SOLVAY); WO 94/02618 (GIST-BROCADES N.V.); and WO 96/34946 (NOVO NORDISK A/S).
However, even though a number of useful protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses.
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
The present inventors have intensively studied numerous of the possible combinations of the N252, T255 and S259 residues of SAVINASE(copyright), and identified a number of variants with increased improved wash performance.
For further details reference is made to working examples herein (vide infra).
Accordingly, the present invention relates in its first aspect to a subtilase protease variant having improved wash performance in detergents, comprising modification(s) in position(s) 252, 255 and/or 259.
Preferably a subtilase variant according to the invention comprises modifications in both positions 252 and 255, and more preferred comprises modifications in all three positions 252, 255, and 259.
In a second aspect the invention relates to a subtilase enzyme variant having improved wash performance in detergents, comprising at least one modification chosen from the group comprising:
252L+255I
252V+255A
252M+255C+259H
252S+255E+259C
252K+255S+259C; or
a variant comprising one or more conservative modification(s) in any of the above mentioned variants (e.g. a conservative modification of a 252L(hydrophobic a.a.)+255I variant include variants such as 252I(hydrophobic a.a.)+255I, and 252V (hydrophobic a.a.)+255I.
In a third aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the invention.
In a fifth aspect the invention relates to a microbial host cell transformed with an expression vector according to the fourth aspect.
In a further aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product.
Even further the invention relates to a composition comprising a subtilase variant of the invention.
Finally the invention relates to the use of the mutant enzymes for a number of industrial relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.
Prior to discussing this invention in further detail, the following term will first be defined.
In describing the various enzyme variants produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)
According to this the substitution of Glutamic acid for glycine in position 195 is designated as:
Gly 195 Glu or G195E
a deletion of glycine in the same position is:
Gly 195* or G195*
and insertion of an additional amino acid residue such as lysine is:
Gly 195 GlyLys or G195GK
Where a deletion in comparison with the sequence used for the numbering is indicated, an insertion in such a position is indicated as:
*36 Asp or *36D
for insertion of an aspartic acid in position 36
Multiple mutations are separated by pluses, i.e.:
Arg 170 Tyr+Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).
Numbering of Amino Acid Positions/residues
If no other mentioned the amino acid numbering used herein correspond to that of the subtilase BPN{grave over ( )} (BASBPN) sequence. For further description of the BPN{grave over ( )} sequence see Siezen et al., Protein Engng. 4 (1991) 719-737 and FIG. 1.
Serine Proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease-produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases have been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. and FIG. 1 herein.
One subgroup of the subtilases, I-S1, comprises the xe2x80x9cclassical xe2x80x9d subtilisins, such as subtilisin 168, subtilisin BPNxe2x80x2, subtilisin Carlsberg (ALCALASE(copyright), NOVO NORDISK A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL(copyright), Gist-Brocades NV), subtilisin 309 (SAVINASE(copyright), NOVO NORDISK A/S), subtilisin 147 (ESPERASE(copyright), NOVO NORDISK A/S), and alkaline elastase YaB.
xe2x80x9cSAVINASE(copyright)xe2x80x9d
SAVINASE(copyright) is marketed by NOVO NORDISK A/S.
It is subtilisin 309 from B. Lentus and differs from BABP92 only in having N87S (see FIG. 1 herein).
Parent Subtilase
The term xe2x80x9cparent subtilasexe2x80x9d is a subtilase defined according to Siezen et al. (Protein Engineering 4:719-737 (1991)). For further details see description of xe2x80x9cSUBTILASESxe2x80x9d immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modification have been made while retaining the characteristic of a subtilase.
Alternatively the term xe2x80x9cparent subtilasexe2x80x9d may be termed xe2x80x9cwild-type subtilasexe2x80x9d.
Modification(s) of a Subtilase Variant
The term xe2x80x9cmodification(s)xe2x80x9d used in connection with modification(s) of a subtilase variant as discussed herein is defined to include chemical modification as well as genetic manipulation. The modification(s) can be by substitution, deletion and/or insertions in or at the amino acid(s) of interest.
Subtilase Variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
Homologous Subtilase Sequences
Specific amino acid residues of SAVINASE(copyright) subtilase are identified for modification herein to obtain a subtilase variant of the invention.
However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to that of SAVINASE(copyright).
In order to identify other homologous subtilases, within the scope of this invention, an alignment of said subtilase(s) to a group of previously aligned subtilases is performed keeping the previous alignment constant. A comparison to 18 highly conserved residues in subtilases is performed. The 18 highly conserved residues are shown in table I (see Siezen et al. for further details relating to said conserved residues).
After aligning allowing for necessary insertions and deletions in order to maintain the alignment suitable homologous residues are identified. Said homologous residues can then be modified according to the invention.
Using the CLUSTALW (version 1.5, April 1995) computer alignment program (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) Nucleic Acids Research, 22:4673-4680.), with GAP open penalty of 10.0 and GAP extension penalty of 0.1, using the BLOSUM30 protein weight matrix, alignment of a given subtilase to a group of previously aligned subtilases is achieved using the Profile alignments option in the program. For a given subtilase to be within the scope of the invention, preferably 100% of the 18 highly conserved residues should be conserved. However, alignment of greater than or equal to 17 out of the 18 residues, or as little as 16 of said conserved residues is also adequate to identify homologous residues. Conservation of the, in subtilases, catalytic triad Asp32/His64/Ser221 should be maintained.
The previously defined alignment is shown FIG. 1, where the percent identity of the individual subtilases in this alignment to the 18 highly conserved residues are shown too.
Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases and corresponding homologous residues, which can be modified according to the invention. To illustrate this table II below shows a limited list a homologous subtilases and corresponding suitable residues to be modified according to the invention.
It is obvious that a similar or larger table covering other homologous subtilases may easily be produced by a person skilled in the art.
Wash Performance
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, washability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.
Isolated DNA sequence
The term xe2x80x9cisolatedxe2x80x9d, when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5xe2x80x2 and 3xe2x80x2 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term xe2x80x9can isolated DNA sequencexe2x80x9d may alternatively be termed xe2x80x9ca cloned DNA sequencexe2x80x9d.
Isolated protein
When applied to a protein, the term xe2x80x9cisolatedxe2x80x9d indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. xe2x80x9chomologous impuritiesxe2x80x9d (see below)). It is preferred to provide the protein in a highly purified form, i.e., greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term xe2x80x9cisolated proteinxe2x80x9d may alternatively be termed xe2x80x9cpurified proteinxe2x80x9d.
Homologous Impurities
The term xe2x80x9chomologous impuritiesxe2x80x9d means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originate from the homologous cell where the polypeptide of the invention is originally obtained from.
Obtained From
The term xe2x80x9cobtained fromxe2x80x9d as used herein in connection with a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell in which a gene from the source have been inserted.
Substrate
The term xe2x80x9cSubstratexe2x80x9d used in connection with a substrate for a protease is should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Product
The term xe2x80x9cproductxe2x80x9d used in connection with a product derived from a protease enzymatic reaction should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.